Grantee Research Project Results
2011 Progress Report: Comparison of Gnotobiotic and Conventional Mice for Predicting the Allergenic Potential of Proteins Introduced into Genetically Engineered Plants
EPA Grant Number: R834824Title: Comparison of Gnotobiotic and Conventional Mice for Predicting the Allergenic Potential of Proteins Introduced into Genetically Engineered Plants
Investigators: Baumert, Joseph L , Goodman, Richard E. , Peterson, Daniel H
Institution: University of Nebraska at Lincoln
EPA Project Officer: Aja, Hayley
Project Period: September 15, 2010 through September 14, 2013 (Extended to September 14, 2014)
Project Period Covered by this Report: September 15, 2010 through September 14,2011
Project Amount: $423,546
RFA: Approaches to Assessing Potential Food Allergy from Genetically Engineered Plants (2009) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The proposed research focuses on the development of a more reliable, practical and predictive animal model that can be used to evaluate the allergenic potential of proteins introduced into genetically engineered plants. The objectives of this research project are to: (1) evaluate sensitization responses in germ-free mice and mice having different defined intestinal microflora, to orally presented purified proteins (potent allergen, peanut Ara h 2; moderate allergen, egg-white lysozyme and non-allergenic soybean lipoxygenase); (2) evaluate the importance of the food matrix (peanut, egg-white and soybean) on sensitizing potential of pure proteins; and (3) test for differences in absorption (serum concentrations) of sensitizing proteins (Ara h 2, lysozyme and soybean lipoxygenase) in (1) and (2), as a potential source of differences in allergic sensitization.
Progress Summary:
Future Activities:
Protein purification and characterization/identification will continue in year 2 of the grant to obtain sufficient quantities of peanut Ara h 2 and soybean lipoxygenase if needed. The addition of the AKTA Avant FPLC instrument in Dr. Baumert’s laboratory will aid in increasing the efficiency and repeatability of protein purification from natural food sources. All purified proteins will be analyzed using SDS-PAGE analysis, immunoblot analysis when antibodies directed against the protein of interest are available, and confirmatory tandem mass spectrometry analysis will be conducted. The purified proteins will be used for oral sensitization of conventional, germ-free and mono-associated mice of various strains. Breeding pairs of germ-free C3H/HeN mice will be purchased from INRA in France (from Dr. Sylvie Rabot). These mice will be transported to our facilities at the University of Nebraska at Lincoln (UNL) at the beginning of 2012, allowed to acclimate for 3-4 weeks, and bred to develop a germ-free line of C3H/HeN mice at UNL. Once a suitable population of germ-free mice are developed (anticipated by July 2012), sensitization experiments on the germ-free mice will commence using BLG, Ara h 2, HEL, and soybean lipoxygenase as sensitizing proteins. A portion also will be mono-associated with B. fragilis and evaluated for sensitization and allergic response to the outlined proteins. Work on the B cell ELISPOT will continue to evaluate this methodology to detect specific IgE secreting B cells from lymphocytes of mice sensitized to Ara h 2, BLG, and soybean lipoxygenase. As the mice from these experiments are euthanized, sera will be collected to determine if digestion resistant peptides of these allergenic proteins can be measured with both ELISA methods and by tandem mass spectrometry as outlined in Objective 3 of the proposal.
Journal Articles:
No journal articles submitted with this report: View all 10 publications for this projectSupplemental Keywords:
Anaphylaxis, antibody, GM, risk assessment, sensitive population, human health risk, food allergenicity, oral allergy syndrome, allergic sensitization;Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.